Methods and compositions for the reduction of neutrophil influx and for the treatment of bronchpulmonary dysplasia, respiratory distress syndrome, chronic lung disease, pulmonary fibrosis, asthma and chronic obstructive pulmonary disease

ABSTRACT

The present invention relates generally to the use of recombinant human CC10 (rhCC10), also known as recombinant human uteroglobin, for use as a therapeutic in the treatment of Respiratory Distress Syndrome (RDS), Bronchopulmonary dysplasia (BPD), chronic lung disease and/or pulmonary fibrosis, Asthma and Chronic Obstructive Pulmonary Disease (COPD). More particularly, the invention provides methods, including broadly the critical dosage ranges of rhCC10, which may be administered to safely and effectively treat the aforementioned conditions. The invention further provides a composition useful in administering rhCC10 to humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of a application Ser. No. 11/378,798,filed Mar. 16, 2006, which is a continuation-in-part of application Ser.No. 11/189,229, filed Jul. 25, 2005, which is a continuation-in-part ofapplication Ser. No. 09/835,784, filed Apr. 13, 2001, now abandoned,which is a continuation-in-part of application Ser. No. 09/549,926,filed Apr. 14, 2000, now abandoned, which is a continuation-in-part ofapplication Ser. No. 09/120,264, filed Jul. 21, 1998, now abandoned,which is a continuation-in-part of application Ser. No. 09/087,210,filed May 28, 1998, now abandoned, which is a continuation-in-part ofapplication Ser. No. 08/864,357, filed May 28, 1997, U.S. Pat. No.6,255,281. Each of the aforementioned applications and patent areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods of reducing the influx ofneutrophils into the lungs of a human. More specifically the presentinvention relates to methods of treating respiratory distress syndrome(RDS) bronchopulmonary dysplasia (BPD), chronic lung disease, pulmonaryfibrosis, asthma and chronic obstructive pulmonary disease (COPD) inhumans and compositions useful for the same. Yet more specifically, thepresent invention relates to methods of treating the above usingrecombinant human CC10 and compositions useful for the same.

BACKGROUND

The influx of neutrophils into the lung is known to be a cause of thedestruction of functional lung tissue and the harmful symptoms of RDS,BPD, chronic lung disease, pulmonary fibrosis, asthma and COPD.Neutrophil influx is the migration of neutrophils from the blood intotissue in response to any type of irritation or injury to the tissue. InRDS, BPD, chronic lung disease and/or pulmonary fibrosis, asthma andCOPD total cell influx and neutrophil influx results in damage to anddestruction of pulmonary tissue, ultimately causing functional lungtissue to be replaced with non-functioning fibrotic tissue. Thus,neutrophil influx is ultimately responsible for causing pulmonaryfibrosis to the lungs, which lead to damaged lung tissue and possiblydeath.

When circulating neutrophils are activated by chemical and cytokinesignals released by damaged or irritated tissue, for example, by IL-8from the lungs, they adhere to the walls of blood vessels in the damagedtissue, following the chemical and cytokine signal to the source, andmigrate through the vascular endothelia and into the damaged lung.Neutrophils release many powerful enzymes, such as myeloperoxidase(MPO), an enzyme that chemically damages and modifies all proteins inits local vicinity, usually inactivating them. MPO damages local lungtissue and proteins, as well as those of the infectious agents.Neutrophils also release powerful proteases, such as elastase, thatindiscriminately degrade host and pathogen proteins alike. Thus,activated neutrophils that migrate into the lungs in response to someirritation of the respiratory tract release non-specific destructiveenzymes that damage the host's respiratory tissues, as well as anyinfectious agents present. These, and other, powerful non-specificdestructive mediators released by neutrophils damage all cell types anddestroy lung tissue, including breaking down the bronchiolar andalveolar structure, resulting in decreased lung function and respiratorydistress. Local vascular structure is also damaged, resulting inincreased vascular permeability and leakage of serum proteins into thelung and tracheal fluid, further impairing function.

This non-speciflc neutrophil response results in greater damage torespiratory tissue than the original irritant or infectious agent.Patients that develop respiratory symptoms or respiratory distress as aresult of an exposure to an irritant such as an allergen, air-borneparticulate matter, chemicals, or infectious agents often experience theworst symptoms after the irritant or infectious agent is cleared fromthe respiratory tract. Neutrophils are substantially responsible forthis over-reaction.

Neutrophil influx to the lungs is measured in patients by counting thenumber of neutral-staining white cells per unit volume in trachealfluids (referred to as tracheal aspirate fluid or TAF). Tracheal fluidsare continuous with bronchial fluids, alveolar fluids, and nasal andsinus fluids. Tracheal fluid composition is representative of pulmonaryfluids in the lower respiratory tract, as well as the upper respiratorytract (nasal and sinus fluid).

Cytokines, like IL-6 and IL-8, are released by local epithelial cells,endothelial cells, and fibroblasts. The levels of cytokines, for exampleIL-6 and IL-8, are measured in lung fluids such as TAF and in plasma orserum. Cytokines are basic regulators of all neutrophil functions. Undernormal conditions, neutrophils move along microvascular walls via lowaffinity interaction of selectins with specific endothelial carbohydrateligands. However, during the inflammatory response, chemotactic factorsand proinflammatory cytokines signal the recruitment of neutrophils(neutrophil influx) to sites of infection and/or injury. Neutrophilsthen penetrate the endothelial layer and migrate through connectivetissue to sites of injury, for example the lungs, where they accumulateand adhere to extracellular matrix components such as fibronectin and/orcollagen.

RDS affects 10% of all premature infants and only rarely affects thoseborn at full-term. RDS also affects adults. The disease is caused by alack of lung surfactant, a chemical that normally appears in maturelungs, or by tissue damage to the lungs from being on a mechanicalventilator and oxygen for a significant amount of time. Surfactant keepsthe air sacs from collapsing and allows them to inflate with air moreeasily. In respiratory distress syndrome, the air sacs collapse andprevent the child from breathing properly. In infants, symptoms usuallyappear shortly after birth and become progressively more severe. Ifsymptoms of RDS persist, the condition is considered BPD if a baby isdependent on artificially supplied oxygen at 36 weeks' postconceptionalage (PCA—also known as post-conceptual age). In a child or adult, ifsymptoms of RDS persist, the condition is considered chronic lungdisease and/or pulmonary fibrosis if the patient is dependent onartificially supplied oxygen following a respiratory distress episode.

BPD affects 20-60% of all premature, very low birth weight infants. BPDand RDS are associated with substantial morbidity and mortality as wellas extremely high health care costs. Although the widespread use ofintratracheally administered exogenous surfactant and antenatal steroidtherapy has reduced the overall severity of BPD, the prevalence of thiscondition has increased with improved survival of very low birth weightinfants, BPD is a multi-factorial disease process that is the end resultof an immature, surfactant deficient lung that has been exposed tohyperoxia, mechanical ventilation and infection. Furthermore, it is welldocumented that increased concentrations of cytokines and cells presentin the tracheal aspirate fluid of premature infants within the first fewdays of life are associated with the subsequent development of RDS andBPD. Still further it is known that higher levels of fibronectin arepresent in the tracheal fluid and lungs of patients suffering from RDSand BPD and thus it causes and contributes to respiratory distress.Thus, treating and preventing RDS and BPD by providing improved lungfunction during the first few days of life of a premature infant iscritical to the long term survivability of the infant.

Asthma is a chronic lung condition characterized by difficulty inbreathing. Symptoms include: wheezing, coughing shortness of breath andchest tightness. People with asthma have extra sensitive orhyperresponsive airways. The airways react by narrowing or obstructingwhen they become irritated. This makes it difficult for the air to movein and out. This narrowing or obstruction causes the symptoms of asthma.The narrowing or obstruction of the airways is caused by: airwayinflammation (meaning that the airways in the lungs become red, swollenand narrow) or bronchoconstriction (meaning that the muscles thatencircle the airways tighten or go into spasm)

COPD is a lung disease in which the lungs are damaged, making it hard tobreathe. In COPD, the airways are partly obstructed, making it difficultto get air in and out of the lungs. Most cases of chronic obstructivepulmonary disease (COPD) develop after repeatedly breathing in fumes andother things that irritate and damage the lungs and airways, for exampleby smoking. The lungs and airways are highly sensitive to irritants.They cause the airways to become inflamed and narrowed, and they destroythe elastic fibers that allow the lung to stretch and then return to itsresting shape. This makes breathing air in and out of the lungs moredifficult. COPD may also be caused by a gene-related disorder calledalpha 1 antitrypsin deficiency. Alpha 1 antitrypsin is a protein thatinactivates destructive proteins. People with antitrypsin deficiencyhave low levels of alpha 1 antitrypsin; the resulting imbalance ofproteins leads to the destruction of the lungs and COPD.

Symptoms common to RDS, BPD, chronic lung disease, pulmonary fibrosis,asthma and COPD include respiratory insufficiency (i.e. lungs unable toadequately oxygenate the blood and remove carbon dioxide), increasedairway resistance, inflammation and fibrosis of the lungs. Each of thesesymptoms are substantially caused by excessive levels of neutrophils,IL-6, IL-8, and total cells in the tracheal fluid. Excess total proteinin the tracheal aspirate fluid (“TAF”) or bronchoalveolar lavage fluid(“BAL”) is also associated with lung inflammation and fibrosis in MDS,BPD, chronic lung disease, pulmonary fibrosis, asthma and COPD.

Glucocorticoids, also known as corticosteroids, are powerfulanti-inflammatory agents that are known to improved lung function, toreduce the incidence of BPD in premature infants, and to improve thesymptoms of RDS, BPD, chronic lung disease and/or pulmonary fibrosis,asthma and COPD. However, they are not completely safe to use. There aredangerous, often life-threatening side effects associated with the useof glucocorticoids in infants, children and adults. In infants,corticosteroids are avoided in clinical practice because they causegrowth retardation, disproportionate growth inhibition of the centralnervous system and head, and severe neurological impairment. Inchildren, normal growth is stunted, resulting in small stature, due totreatment with corticosteroids. And in adults, cardiovascularcomplications, including hypertension and stroke, are major side effectsof corticosteroids. In all patients, corticosteroids lower the patient'simmune function and leave them susceptible to infection of all types(bacterial, viral, fungal, etc.), sometimes resulting in a lethalinfection. Thus, safety is a major consideration in the choice ofanti-inflammatory agent used to treat, prevent or cure RDS, BPD, chroniclung disease and/or pulmonary fibrosis, asthma and COPD and theirrelated respiratory symptoms, reduce the severity of asthma or allergy,and prevent the progression of existing chronic lung disease such asCOPD or the development of chronic lung disease such as BPD. It is asignificant challenge to find an anti-inflammatory agent powerful enoughto alleviate respiratory symptoms and which is safe to use.

Human CC10 (hereinafter CC10), also known as uteroglobin, is a smallhomodimeric secretory protein produced by mucosal epithelial cells. Inhumans, Clara cells, a type of mucosal epithelial cell located in theairways, are the main site of CC10 production. CC10 also circulates inthe blood and is excreted in urine. CC10 is known to haveanti-inflammatory properties. CC10 inhibits secretory PLA₂, an enzymethat degrades surfactant and facilitates eicosanoid biosynthesis.Eicosanoids are a family of lipdphilic compounds includingprostaglandins, leukotrienes, thromboxanes, and other arachidonic acidmetabolites.

Further information concerning rhCC10, its structure and methods of useis found in U.S. Pat. No. 6,255,281, its continuation-in-part, U.S.patent application Ser. No. 09/087,210, and in the following U.S. PatentApplication Publication Nos.: US 2002-0160948, US 2002-0160948, US2003-0008816, US 2003-0109429, US 2003-0207795, US 2002-0173460, US2002-0169108, US 2005-0261180, US 2004-0047857, and US 2006-0025348, allof which are incorporated by reference in their entirety.

Very low concentrations of CC10 have been found in the TAF or BAL ofpatients suffering from RDS, Asthma and COPD and BPD. For example, verylow concentrations of CC10 have been found in the tracheal aspiratefluid (TAF) of ventilated premature infants suffering from BPD relativeto normal levels. These infants are not yet able to produce enoughnatural CC10 on their own, and develop severe lung inflammation.Normally, the appearance of CC10 in the amniotic fluid dates from 16weeks of gestation and increases as a function of gestational as well aspostnatal age. CC10 concentrations in tracheal fluid, measured bydetermining the amount of CC10 protein in the tracheal aspirate fluid,of premature infants born at 28-32 weeks of gestation have been found tobe 2-4 orders of magnitude less than those found in the tracheal sputum(a.k.a. tracheal fluid) of healthy adults. CC10 concentrations correlatein a negative fashion with the concentration of inspired oxygen requiredby preterm infants with BPD. That is, infants with lower CC10 in TAFrequire greater amounts of supplemental oxygen. In fact, not only areCC10 concentrations lower in tracheal fluid from infants who either diedor developed BPD, but the limited amount of available CC10 was oxidizedand demonstrated less immunoreactivity relative to controls.

Recombinant CC10 (recombinant human CC10) has not been previously usedto treat patients, including preterm infants for a number of reasons.First, rhCC10 of sufficient purity has not been previously available.Nor was it known whether rhCC10 caused specific toxicity or triggered animmune response to endogenous CC10 when administered. Furthermore, CC10is known to inhibit platelet aggregation, thus negatively impacting theability of the blood to clot. CC10 is also known to suppress the immunesystem, which could lead to adverse patient consequences, renderrecipients more susceptible to infection, and prohibit its use inhumans, including premature infants. It was not known what dosage ordosage range would avoid deleterious immunogenicity, specific toxicity,and inhibition of platelet aggregation and suppression of the immunesystem.

Furthermore, it was not known whether rhCC10 would cause significantlylower total protein concentrations in the tracheal fluids of patients orwhat dosage to administer to achieve significantly lower total proteinconcentrations in the tracheal fluid of patients, a necessary outcome intreating BPD.

Additionally, it was not known whether rhCC10 would cause significantlylower total cell, neutrophil, IL-6 or IL-8 levels in patients or whichdosage to administer to achieve significantly lower neutrophil, IL-6 orIL-8 levels in patients, also a necessary outcome in treating RDS, BDP,chronic lung disease and/or pulmonary fibrosis, Asthma, and COPD.

As shown below, the prior technological difficulties in using CC10 toprovide a safe, well-tolerated and effective treatment for RDS, BDP,chronic lung disease, pulmonary fibrosis, asthma, and COPD have beenovercome.

OBJECTS OF THE INVENTION

The foregoing provides a non-exclusive list of the objectives achievedby the present invention:

It is a primary object of the invention to treat, cure or prevent RDS,BDP, chronic lung disease and/or pulmonary fibrosis, asthma, and COPD inhumans.

It is a further object of the invention to treat cure or prevent RDS,BDP, chronic lung disease and/or pulmonary fibrosis, asthma, and COPD inhumans by reducing total cell counts, neutrophil counts, total proteinconcentration IL-6 levels and/or IL-8 levels in the serum or trachealfluid, and therefore the lungs, of patients.

It is a further object of the invention to provide a safe,well-tolerated and effective dosage range which accomplishes the aboveobjectives and does not significantly inhibit platelet aggregation,suppress the immune response or increase the frequency or severity ofadverse events.

It is yet another object of the invention to provide a safe,well-tolerated and effective dosage which provides a substantiallyeffective range of CC10 levels in patient serum, tracheal fluid andurine.

SUMMARY OF THE INVENTION

These and other objects, features and advantages are achieved byadministering rhCC10 in a dosage range given at appropriate intervals,or in one dose to treat, cure or prevent RDS, BDP, chronic lung disease,pulmonary fibrosis, asthma, and COPD.

These and other objects, features and advantages are also achieved byadministering rhCC10 in a dosage range given at appropriate intervals orin one dose where a patient shows one or more of the following: IL-6levels below 200 pg/ml of tracheal aspirate fluid, IL-8 levels below 100pg/ml in serum, total neutrophil cell counts below 20 cells×10⁴/ml oftracheal aspirate fluid, total cell counts below 50 cells×10⁴/ml oftracheal aspirate fluid, and total protein concentration below 400 μg/mlof tracheal aspirate fluid, such dosing being continued until the RDS,BDP, chronic lung disease and/or pulmonary fibrosis, asthma, or COPD hasbeen treated, cured, or prevented.

These and other objects, features and advantages are also achieved byadministering rhCC10 such that it does not inhibit platelet aggregation,suppress the immune response or increase the frequency or severity ofadverse events.

In certain aspects of the invention, rhCC10 is administeredintratracheally in a dose between about 1.5 and about 5.0 mg/kg ofpatient body mass or in multiple doses which taken together achieve thisdosage range to treat, cure or prevent RDS, BDP, chronic lung diseaseand/or pulmonary fibrosis, asthma, or COPD. In another aspect, an rhCC10dose or doses adding up to between about 1.5 and about 5.0 mg/kg ofpatient body mass may be repeated at appropriate intervals to treat,cure or prevent RDS, BDP, chronic lung disease and/or pulmonaryfibrosis, asthma, or COPD. In yet other aspects of the invention, rhCC10is administered intratracheally in accordance with the above aspects butin a dose or doses adding up to between about 15 nanograms/kg of patientbody mass and about 10 mg/kg of patient body mass or in a dose adding upto between about 0.15 mg/kg and about 5 mg/kg of patient body mass.Whether administered intratracheally or otherwise, rhCC10 may be givenalone, in conjunction with, before or after surfactant therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph of the CC10 concentration in the tracheal aspiratefluid of patients over time that were given placebo, rhCC10 at 1.5 mg/kgof body mass or rhCC10 at 5 mg/kg of body mass.

FIG. 2 is a bar graph of the CC10 concentration in the serum of patientsover time that were given placebo, rhCC10 at 1.5 mg/kg of body mass orrhCC10 at 5 mg/kg of body mass.

FIG. 3 is a bar graph of the CC10 concentration in the urine of patientsover time who were given placebo, rhCC10 at 1.5 mg/kg of body mass orrhCC10 at 5 mg/kg of body mass.

FIG. 4 is a bar graph of total cell counts in the tracheal aspiratefluid of patients over time who were given placebo, or rhCC10 at 5 mg/kgof body mass.

FIG. 5 is a bar graph of total neutrophil counts in the trachealaspirate fluid of patients over time who were given placebo or rhCC10 at5 mg/kg of body mass.

FIG. 6 is a bar graph of total protein concentration in the trachealaspirate fluid of patients over time who were given placebo, rhCC10 at1.5 mg/kg of body mass or rhCC10 at 5 mg/kg of body mass.

FIG. 7 is a bar graph of IL-6 levels in the tracheal aspirate fluid ofpatients over time who were given placebo, rhCC10 at 1.5 mg/kg of bodymass or rhCC10 at 5 mg/kg of body mass.

FIG. 8 is a bar graph of IL-8 levels in the serum of patients at 48hours after administration who given placebo, rhCC10 at 1.5 mg/kg ofbody mass or rhCC10 at 5 mg/kg of body mass.

DETAILED DESCRIPTION

The present invention relates to the critical dosages and timing ofadministration of rhCC10 to treat, cure or prevent RDS, BDP, chroniclung disease and/or pulmonary fibrosis, asthma, and COPD in humans.rhCC10 is preferably obtained by the processes described in U.S. PatentApplication Publication Nos. US 2003-0109429 and US 2003-0207795, bothof which are incorporated by reference in their entirety, or via anyother process which yields pharmacutical grade rhCC10. The rhCC10 of theembodiments of the present invention may also be administered by theintratraceal, endotracheal, dialysate, ophthalmic, intravenous,systemic, or oral routes. Furthermore the rhCC10 of the embodiments ofthe present invention may be administered with, without, before or aftersurfactant therapy.

Preferably, in treating or preventing RDS or BPD rhCC10 is administeredduring the first day of an infant patient's life. More preferably,rhCC10 is administered as soon as medically possible during the firstday of an infant patient's life, for example, and without limitation,within about 30 minutes of intubation and receipt of surfactant.

Premature infants are typically intubated for the purposes ofadministering oxygen and inflating their lungs, which would collapsewithout intubation. Intubation can then serve as a direct route for theintratracheal administration (also known as endotracheal administration)of medicines, such as surfactants, to the lungs. Thus the preferredroute of administration for rhCC10 is also intratracheal administration,however alternate routes of administration are also possible such asinhalation, inhalation of pegylated rhCC10, injection into the muscletissues, intravenous injection, intranasal administration, oraladministration and administration by suppository.

rhCC10 may also be administered to treat, cure or prevent chronic lungdisease and/or pulmonary fibrosis, asthma, and COPD. Based on theresults described herein, rhCC10 will have therapeutic benefit topatients suffering from chronic lung disease and/or pulmonary fibrosis,asthma, and COPD. More specifically, and as shown below, rhCC10, whendosed at the amounts described below, lowers neutrophil counts, IL-6levels and IL-8 levels in humans, and thus will provide an effectivetreatment RDS, Asthma and COPD.

One method of measuring the therapeutic effect of rhCC10 on a patient isto measure conditions present in the tracheal aspirate fluid, which isindicative of conditions present in the tracheal and bronchoalveolarfluid of the patient's lungs. Such conditions may be one or more of thefollowing: IL-6 levels, IL-8 levels, total neutrophil counts, total cellcounts and total protein concentration.

A method of measuring the concentration of CC10 in a patient, and thusdetermining whether treatment has established therapeutically effectivelevels of CC10 in the patient, may include one or more of following:measuring CC10 concentration in TAF, serum or urine.

With reference to the following embodiments, rhCC10 may be administeredto achieve certain desired effects, establishing that therapeuticallyeffective levels of CC10 have been achieved in the patient, while at thesame time avoiding other deleterious effects. For example, rhCC10 may beadministered to achieve concentrations of CC10 in the tracheal aspiratefluid which exceed the deficient production of endogenous CC10 by thepremature infant. rhCC10 may also be administered to achieve early peakserum concentrations of rhCC10 in patients, for example within about 6hours after administration. As a further example, when administered inaccordance with the described methods, rhCC10 administration may alsoachieve a peak concentration in urine at about 12 hours for example. Asyet another example, rhCC10 may be administered so as to achievesignificantly lower cell counts, neutrophil cell counts, proteinconcentrations, IL-6 levels and IL-8 levels in the patient's trachealaspirate fluid and in the patient's lungs.

Furthermore, rhCC10 may be administered such that it does notsignificantly reduce the patient's endogenous CC10 production, inhibitplatelet aggregation or cause an adverse immunologic reaction.

To effectuate the desired outcomes which are further described below,reference is made to methods of administration described in thefollowing embodiments:

In one embodiment, a dose or multiple doses of rhCC10 equaling a doseranging from about 1.5 to about 5 mg/kg of body mass may beadministered. In another embodiment a dose or multiple doses of rhCC10equaling a dose ranging from about 15 nanograms/kg of body mass to about10 mg/kg of body mass is administered. In still another embodiment adose or multiple doses of rhCC10 equaling a dose ranging from about 0.15mg/kg and about 5 mg/kg of patient body mass is administered.

In yet another embodiment the above doses of rhCC10 may be administeredintratracheally to the patient. In yet another embodiment, the abovedoses of rhCC10 may be administered to the patient by aerosol. In afurther embodiment rhCC10, in accordance with the methods describedabove, may be administered prior to, during or after surfactant therapy.In still another embodiment, rhCC10, in accordance with the methodsdescribed above, may be administered to treat RDS, BPD, asthma, chroniclung disease, pulmonary fibrosis or COPD in a patient.

The doses of rhCC10 described above may be administered daily, more thanonce daily, every other day or in a tapered fashion depending upon theseverity of disease being treated, the patient's overall health, andwhether an acute or chronic condition is being treated. For example, themore severe the disease condition, a higher the amount of rhCC10 wouldbe required to effectively treat the disease. For maintenance therapy ofchronic disease, for example, to prevent an exacerbation of chronicAsthma, RDS, COPD, BDP or other pulmonary condition, lower doses wouldbe required. It is understood that a physician would be able to monitorand adjust doses as needed based on the patient's symptoms and responsesto therapy and within the parameters and dose ranges described in theembodiments of the present invention.

The following detailed examples are illustrations of embodiments. Itshould be clear that these are not intended to limit the scope of thepresent invention.

EXAMPLE 1 Administration of rhCC10 to Premature Infants

Patients were enrolled in a placebo-controlled, blinded, dose rangingstudy at four hospital sites.

rhCC10 was produced in E. coli bacteria and purified by a proprietaryprocess (Claragen, Inc., College Park, Md.), described in U.S.Application Publication Nos. US 2003-0109429 and US 2003-0207795, bothof which are incorporated by reference in their entirety. The proteinfor the study was provided as a >98% pure solution of recombinant humanCC10 homodimer. The biological activity of each batch was compared usinga proprietary secretory PLA₂ inhibition assay, described in U.S.Application Publication Nos. US 2002-0169108 which is incorporatedherein by reference.

Newborn infants who met the following criteria were enrolled: 1) age ≦24h; 2) birth weight between 700 and 1,300 g; 3) gestational age ≧24 wk;4) diagnosis of RDS based on clinical and radiographic criteria; 5)requirement for intubation and mechanical ventilation; 6) receipt ofsurfactant, 100 mg/kg (Survanta, Ross Laboratory). Patients could begiven subsequent doses of surfactant if clinically indicated followingrhCC10 administration. Table 1 depicts the composition of the studygroups (cohorts):

TABLE 1 Study Population Gestational Birth Race Any Age Weight Sex(White/Black/ Maternal (weeks) (grams) (Male/Total) Hispanic/Asian)Steroids Placebo 26.5 ± 1.6 943 ± 137 5/7 (71%) 3, 2, 1, 1 7/7 1.5 mg/kg27.6 ± 1.2 981 ± 159 3/7 (43%) 6, 2, 0, 0 6/7 5.0 mg/kg 26.5 ± 1.2 878 ±205 3/6 (50%) 4, 2, 1, 0 6/6

rhCC10, was formulated in a volume of 2 ml/kg of sterile, unbufferedsaline. Patients were enrolled in two cohorts, each comparing study drugto placebo. The first cohort consisted of 12 patients, randomized sothat one-third received placebo and the other two-thirds received rhCC10at 1.5 mg/kg of body mass of the study drug. After the first cohort ofpatients was enrolled and the safety data reviewed by the DSMC, a secondcohort of 12 patients was enrolled. They were also randomized so thatone-third received placebo and the other two-thirds received rhCC10 at5.0 mg/kg of body mass.

Each patient then received a single dose of the study drug (or placebo)as soon as possible after surfactant replacement therapy, but not longerthan 4 h after surfactant. Study drug or placebo was administeredintratracheally (IT) in two equal aliquots via a pre-measured feedingtube placed into the distal third of the endotracheal tube, with thepatient in the right and then left lateral decubitus position and 30° ofTrendelenburg.

As described in greater detail in Examples 2-4, pharmacokinetic analyseswere conducted on samples of TAF, serum, and urine samples and analysesof cells counts and protein levels were performed on samples of TAF.

TAF was obtained by instilling 1 ml of saline into the endotracheal tubeand suctioning the fluid into a Leuken's trap. The catheter was thenwashed with an additional 1 ml saline. In some cases the first trachealaspirate was obtained prior to surfactant administration (baseline).Subsequent TAF collections were obtained at 12, 24, 48 and 72 hourspost-administration. TAF was only collected if infants continued torequire intubation and mechanical ventilation. The TAF was centrifugedat 300×g for 10 minutes to pellet the cells. The supernatant was removedand frozen at −70° C.

The pharmacokinetic analysis was conducted on samples of TAF, serum, andurine samples using a competitive ELISA for human CC10 developed by thesponsor. The assay utilizes a single anti-human CC10 polyclonal antibodyas a capture reagent. CC10 in the sample competes with a syntheticCC10-HRP (horseradish peroxidase) conjugate for antibody binding sitesin the plate wells. Thus, the signal decreases with increasing CC10concentration in the sample. Samples were run in duplicate and astandard curve was run for each set of assays using rhCC10 calibrators.The limit of detection is 5 ng/ml and the results were reproducible withcoefficients of variation typically under 20%. The assay does not appearto distinguish between native and recombinant CC10, thus total CC10levels were measured. Immunogenicity of the study drug was assessed bytitration of anti-C10 antibodies in serum obtained at 28 days postadministration.

Referring to Examples 5-8 below, analyses for pulmonary inflammatorymarkers were performed as follows: The TAF cell pellet was resuspendedand cell counts performed using a hemocytometer. Differential cellcounts in TAF were determined by cytocentrifugation and differentialstaining. Total protein in TAF was measured using the Pierce BCAtechnique, and a panel of cytokines (Multiplex cytokine analysis,Luminetics Corp.) was measured in TAF from all three experimental groupsat 0, 24 and 48 hours post-administration. IL-6 and IL-8 cytokines weremeasured in TAF from patients in all three groups at times 0, 1 and 2days (with a minimum of three and maximum of seven samples/group).

Concentrations of CC10 and analysis of inflammatory markers over timewere examined by using mixed model analysis of variance to test theinteraction of time and dose. Non-parametric testing was performed whenunequal variance was detected. Sample characteristics, the incidence ofcomplications and clinical outcomes were analyzed by Fisher's Exact Testfor categorical variables or one way analysis of variance for continuousvariables.

EXAMPLE 2 TAF Concentrations of CC10 in Patients Treated with rhCC10

With reference to FIG. 1 it has been found that during the first 48hours of life, after an initial dose of rhCC10, significantly increasedoverall CC10 concentration occurred in patients receiving rhCC10 indosages comprising either 1.5 mg/kg of body mass or 5 mg/kg of body massversus placebo. Thus, administration of rhCC10 during the first 24 hoursof life has a significant positive impact on CC10 levels in patientsduring the first two days of life. Furthermore, administration of rhCC10will increase overall CC10 concentrations in patients.

Reference is now made to Table 2, as well as to FIG. 1, the contents ofwhich are further described in this example.

TABLE 2 Average TAF CC10 Concentrations CC10 CC10 CC10 CC10 Conc. inConc. in Conc. in Conc. in TAF** TAF TAF TAF 12 Hours 24 Hours 48 Hours72 Hours Placebo  476 ng/ml  753 ng/ml  916 ng/ml 3435 ng/ml 1.5 mg/kg*2336 ng/ml 1639 ng/ml 2492 ng/ml 1522 ng/ml rhCC10   5 mg/kg* 2400 ng/ml1994 ng/ml 1432 ng/ml  784 ng/ml rhCC10 *dosage units are mg of rhCC10per kg of patient body mass **CC10 concentration in TAF are in units ofng of CC10 per ml of TAF

CC10 concentrations in patients were measured at 12, 24, 48 and 72 hourspost-administration. An average concentration for each patient groupreceiving a particular dose of rhCC10 (1.5 or 5 mg/kg) or placebo wasdetermined as follows. CC10 concentrations in TAF were observed for timepoints where there were at least three patient samples per group (FIG.1). This allowed for analysis of TAF samples for all groups followingadministration of placebo (0.9% sterile saline) or rhCC10 through day 3of life. For safety and logistical reasons, samples were not obtained ifsurfactant had recently been administered or if the infant had beenextubated. At 12 hours of life, CC10 concentrations in TAF from infantstreated with the study drug was significantly higher than the placebogroup, but there was little difference between the two groups whoreceived rhCC10. Over the first 3 days of life, CC10 concentrations inTAF from infants receiving placebo generally increased, whereas CC10levels in treated infants tended to remain constant (1.5 mg/kg of bodymass) or decrease (5 mg/kg of body mass). However, CC10 levels frominfants receiving placebo did not exceed the CC10 levels of thoseinfants receiving rhCC10 in either 1.5 mg/kg of body mass or 5 mg/kg ofbody mass dosages during the first 48 hours of life. Those infantsreceiving 1.5 mg/kg of body mass rhCC10 had the highest levels of CC10at 48 hours.

EXAMPLE 3 Serum Concentrations of CC10 in Patients Treated with rhCC10

Furthermore, with reference to FIG. 2, in one embodiment, rhCC10 may beadministered intratracheally such that peak serum levels of CC10 areachieved within 6 hours of administration. Peak serum levels occurwithin 6 hours, irrespective of the dose of rhCC10 administered. Basedon the results described below and in FIG. 2, peak serum levels willoccur within about 6 hours after administration across all dosageranges.

Reference is now made to Table 3, as well as to FIG. 2, the contents ofwhich are further described in this example.

TABLE 3 Average Serum CC10 Concentrations CC10 CC10 CC10 CC10 CC10 CC10Conc. in Conc. in Conc. in Conc. in Conc. in Conc. in serum** serum**serum** serum** serum** serum** Elimination 0 Hours 6 Hours 12 Hours 24Hours 36 Hours 48 Hours Half-life Placebo 42 ng/ml  43 ng/ml  46 ng/ml 40 ng/ml  37 ng/ml  38 ng/ml Not applicable 1.5 mg/kg* 76 ng/ml 1289ng/ml  782 ng/ml 354 ng/ml 196 ng/ml 101 ng/ml 11.6 hours rhCC10   5mg/kg* 22 ng/ml 2794 ng/ml 1476 ng/ml 798 ng/ml 290 ng/ml 143 ng/ml  9.9hours rhCC10 *dosage units are mg of rhCC10 per kg of patient body mass**CC10 concentration in serum are in units of ng of CC10 per ml of serum

In determining average peak serum levels, blood (0.3 ml) was obtainedfor the measurement of serum concentration of CC10 before drugadministration (0 hours) and at 6, 12, 24, 36, and 48 hours afteradministration of rhCC10. An average concentration for each patientgroup receiving a particular dose of rhCC10 or placebo was determined.

Serum concentrations of CC10 were similar in all 3 groups beforetreatment (FIG. 2). Infants who received rhCC10 had substantially higherserum concentrations than infants receiving placebo and this varied in adose dependent manner. Average peak serum levels after administration ofrhCC10 may range from about 1290 ng/ml of serum to about 2800 ng/mlserum when rhCC10 is given in a single dose of between 1.5 mg/kg of bodymass and 5 mg/kg of body mass. As shown in Table 3, the eliminationhalf-life of a rhCC10 dosage of 1.5 mg/kg of body mass was about 11.6hours, whereas the elimination half-life of a rhCC10 dose of 5 mg/kg ofbody mass was about 9.9 hours. CC10 concentrations in the serum oftreated infants were comparable to placebo levels within 48 hours ofadministration.

EXAMPLE 4 Urine Concentrations of CC10 in Patients Treated with rhCC10

Referring to FIG. 3, in one embodiment, rhCC10 may also be administeredintratracheally at the above-mentioned dosages such that peak CC10levels in urine occur 12 hours after administration. For example, CC10concentrations in the urine of treated infants increase in a largelydose-dependent manner, but are comparable to placebo levels within 48 hof administration.

Reference is now made to Table 4, as well as to FIG. 3, the contents ofwhich are further described in this example.

TABLE 4 Average Urine CC10 Concentrations CC10 CC10 CC10 CC10 Conc. inConc. in Conc. in Conc. in urine** urine** urine** urine** 12 Hours 24Hours 36 Hours 48 Hours Placebo  197 ng/ml  183 ng/ml 180 ng/ml 203ng/ml 1.5 mg/kg* 3312 ng/ml 1226 ng/ml 1513 ng/ml  593 ng/ml rhCC10   5mg/kg* 9239 ng/ml 2613 ng/ml 763 ng/ml 583 ng/ml rhCC10 *dosage unitsare mg of rhCC10 per kg of patient body mass **CC10 concentration inserum are in units of ng of CC10 per ml of urine

Urine samples were obtained at 12, 24, 36 and 48 hours afteradministration of rhCC10. Each urine sample consisted of the totalvolume voided over the previous 12 hours.

In urine, CC10 concentrations in treated infants increased in a largelydose-dependent manner, but were comparable to placebo levels within 48 hof administration (FIG. 3).

EXAMPLE 5 Total Cell Counts in TAF of Patients Treated with rhCC10

As shown in FIG. 4 and in Table 5, total cell counts were performed onTAF fluids and are shown in FIG. 5. Average total cell counts wereobtained by measuring and averaging total cell counts in TAF within theplacebo and 5 mg/kg rhCC10 study groups. Study groups were sampled at0.5, 1, 2, and 3 days post-administration.

TABLE 5 Total Cell Counts in TAF Total cell Total cell Total cell Totalcell count** count** count** count** 0.5 days 1 day 2 days 3 daysPlacebo 1.0 36 61 56 5 mg/kg* 9.8 11 24 14 rhCC10 *dosage units are mgof rhCC10 per kg of patient body mass **total cell counts are in unitsof Cells × 10⁴ per ml of TAF

Total cell counts were significantly lower in the 5 mg/kg group on days1-3 compared to the placebo group. Total cells counts were at leasttwice as low during days 1-3 of life after rhCC10 at 5 mg/kg of bodymass was administered versus placebo.

EXAMPLE 6 Total Neutrophil Counts in TAF in Patients Treated with rhCC10

Total neutrophil counts were performed on TAF fluids in order to gaugerhCC10's effect on inflammation in the lungs and are shown in FIG. 5.Inflammation of the lungs is caused by an excess of neutrophil cellswhich are a cause of RDS, BDP, chronic lung disease, pulmonary fibrosis,asthma, and COPD. Average neutrophil counts were obtained by measuringand averaging neutrophil counts in TAF within each study group (placeboand 5 mg/kg rhCC10). Study groups were sampled at 0.5, 1, 2, and 3 dayspost-administration.

TABLE 6 Total Neutrophil Counts in TAF Total cell Total cell Total cellTotal cell count** count** count** count** 0.5 days 1 day 2 days 3 daysPlacebo 0.1 13.7 31.2 12 5 mg/kg* 2.4 3.4 10.2 4.7 rhCC10 *dosage unitsare mg of rhCC10 per kg of patient body mass **total cell counts are inunits of Cells × 10⁴ per ml of TAF

Neutrophil counts were significantly lower in the 5 mg/kg group relativeto the placebo group. For example, on day two the placebo group'sneutrophil levels were over 30 cells×10(4)/ml of TAF versus about 10cells×10(4)/ml of TAF for the group receiving rhCC10 at 5 mg/kg of bodymass. Therefore, excessive neutrophil cell amounts were minimized in thelungs.

EXAMPLE 7 Total Protein Concentration in TAF of Patients Treated withrhCC10

Referring now to FIG. 6 and to Table 7, total protein levels weremeasured in the TAF of both treatment groups (rhCC10 at 1.5 mg/kg and 5mg/kg of body mass) in order to gauge rhCC10's effect on protein leakand pulmonary edema. Both protein leak and pulmonary edema areconditions damaging to the lungs and symptomatic of RDS, BDP, chroniclung disease, pulmonary fibrosis, asthma, and COPD. Average totalprotein concentrations were obtained by measuring and averaging totalprotein concentrations in TAF within each study group (placebo, 1.5mg/kg rhCC10 and 5 mg/kg rhCC10). Study groups were sampled at 0.5, 1,2, and 3 days post-administration.

TABLE 7 Total Protein Concentrations in TAF Total protein Total proteinTotal protein Total protein conc.** conc.** conc.** conc.** 0.5 days 1day 2 days 3 days Placebo 307 430 536 964 1.5 mg/kg* 565 527 685 264rhCC10   5 mg/kg* 289 334 189 167 rhCC10 *dosage units are mg of rhCC10per kg of patient body mass **total cell counts are in units of μg ofprotein per ml of TAF

Total protein was significantly lower in TAF from both treatment groupscompared to placebo. For example, by day three post-administration,total protein in the TAF of the placebo group was nearly 1000 μg/ml ofTAF whereas total protein in the treatment groups on day three did notexceed 350 μg/ml of TAF. Thus protein leak and pulmonary edema had beenminimized in the treatment groups.

EXAMPLE 8 Total IL-6 Levels in TAF CC10 in Patients Treated with rhCC10

IL-6 cytokine was measured in TAF from patients in all three groups attimes 0, 1 and 2 days post-administration (with a minimum of three andmaximum of seven samples/group).

Referring now to Table 8 and FIG. 7, IL-6 concentrations wereeffectively reduced by the study drug (rhCC10 in saline) in both groups,but increased over time in the placebo group.

TABLE 8 Pharmacokinetic Results: IL-6 Levels in TAF IL-6 IL-6 IL-6 IL-6level level** level** level** 0 days 0.5 days 1 day 2 days Placebo n/a150 291 449 1.5 mg/kg* 572 187 269 99 rhCC10   5 mg/kg* 446 64 103 113rhCC10 *dosage units are mg of rhCC10 per kg of patient body mass**total cell counts are in units of pg of IL-6 per ml of TAF

For example, those patients receiving rhCC10 at 5 mg/kg of body mass hadIL-6 levels below 200 pg/ml of TAF over the first two days followingadministration. Those patients receiving rhCC10 at 1.5 mg/kg of bodymass had IL-6 levels below 300 pg/ml of TAF over the first two dayspost-administration and had an IL-6 level below 100 pg/ml by day two.However, those patients on placebo had steadily increasing IL-6 levels,exceeding 400 pg/ml by day 2. Thus rhCC10, when administered accordingto the present teachings, reverses the upward trend of IL-6 levels inpatient lungs, thus preventing neutrophil influx to the lung.

EXAMPLE 9 Total IL-8 Levels in TAF and Serum in Patients Treated withrhCC10

Referring now to FIG. 8 and Table 9, IL-8 cytokine was measured in TAFfrom patients in all three groups at 48 hours post-administration. IL-8is a potent chemoattractant for neutrophils and other circulatinginflammatory cells, that is released by local epithelial cells, residentimmune cells, and fibroblasts in response to injury or irritation.

TABLE 9 Pharmacological Results: Total IL-8 Levels in Serum IL-8 inSerum** 48 hours Placebo 118 pg/ml  1.5 mg/kg* 89 pg/ml rhCC10   5mg/kg* 72 pg/ml rhCC10 *dosage units are mg of rhCC10 per kg of patientbody mass **total IL-8 levels are in units of pg of IL-8 per ml of serum

IL-8 levels were lower in rhCC10-treated patients than in patientsreceiving placebo. Administration of rhCC10, as depicted above, reducedthe levels of IL-8 released from the lungs and into the systemiccirculation. Thus, this data shows that rhCC10 is effective at treatingor preventing RDS, BDP, chronic lung disease and/or pulmonary fibrosis,asthma, and COPD by lowering IL-8 levels and thus treating the causativeagent of these diseases.

EXAMPLE 10 Outcomes

Table 10 depicts comparative outcomes of patients who received rhCC10versus patients who received placebo. Patients were monitored at sixmonths of corrected age (the developmental timepoint at which they wouldhave been six months old had they been born at the normal 40 weeks ofgestation.) Table 11 depicts further comparative outcomes of patientswho received rhCC10 versus patients who received placebo.

TABLE 10 Outcomes at 6 Month Corrected Age Outcome Placebo 1.5 mg/kg 5mg/kg # with repeat 4/6 4/6 0/5 Respiratory symptoms (cough, wheezing) #with doctor visits 4/6 2/6 3/5 for Respiratory symptoms # hospitalizedfor 3/6 0/6 0/5 breathing problems

Referring to Table 10 above, patients who received rhCC10 therapy hadreduced incidences of respiratory symptoms, e.g. coughing and wheezing.Coughing and wheezing are symptoms common RDS, BDP, chronic lung diseaseand/or pulmonary fibrosis, asthma, and COPD. After receiving rhCC10,patients had fewer doctor visits due to respiratory symptoms, and nopatients were hospitalized for breathing problems in comparison to 50%of infants in the placebo group who were hospitalized for theirrespiratory symptoms. This data shows that rhCC10 significantly reducesthe severity of respiratory symptoms, preventing the need forrehospitalization.

TABLE 11 Observations During Initial Hospitalization Placebo 1.5 mg/kg 5mg/kg Doses of Surfactant 1.9 +/− 10.7 1.4 +/− 0.7 1.5 +/− 0.5 Days onVentilator 12.1 ± 8.6 8.2 ± 7.8 24 ± 13.1 Days on Ventilator   33 ± 12.7 18.7 ± 13.2* 44.3 ± 18.1   and NCPAP Days on O₂  56.6 ± 13.1   49 ±11.2 55 ± 18.1 O₂ at 28 d 7/7 7/7 5/6 O₂ at 36 wk PCA 2/7 1/7 3/6Hospitalized at 36 wk 5/7 (71.4%) 2/7 (28.6%) 4/6 (66.7%) PCA PDA 5/73/8 2/7 Sepsis 0/0 1/8 1/7 IVH 2/7 0/8 1/7 PVL 1/7 0/8 0/7 NCPAP—nasalcontinuous positive airway pressure, NEC—necrotizing enterocolitis;PCA—post-conceptual age, PDA—patent ductus arteriosus,IVH—intraventricular hemorrhage, PVL—periventricular leukomalacia

Referring to Table 11 above, the therapeutic effect of rhCC10 on shortterm respiratory distress is reflected in the decreased requirement foradditional doses of exogenous surfactant therapy. The length of hospitalstays was tabulated for each study group. Only 28.6% of patients in thelow dose group were still hospitalized after 36 weeks compared to 71.4%of patients in the placebo group and 66.7% in the high dose groups.

Patients in the low dose rhCC10 group (1.5 mg/kg of body mass) were onthe ventilator and NCPAP for significantly fewer days than the placebopatients. When total days of mechanical ventilation were evaluated,there was also a trend towards a reduction in the need for ventilatorysupport in the low dose group (the 1.5 mg/kg body mass group).

These results show that rhCC10 therapy, when administered in accordancewith the present teachings, decreased the severity of RDS compared toplacebo and reduced or eliminated the incidence of respiratory problemssevere enough to warrant medical attention or rehospitalization.

Furthermore, this data shows that the safety profile of rhCC10 issuperior to other anti-inflammatory agents such as corticosteroids. Thesafety and tolerability of the study drug were assessed through 36 weeksPCA (Post conceptual age, also known as PMA—post menstrual age) bycomparing the incidence of adverse events in the treatment and placebogroups and to the historical incidence of the adverse events at eachinstitution. No deaths were attributable to administration of rhCC10.

In addition, a preliminary assessment of the efficacy of IT rhCC10 indecreasing the incidence of BPD was made on the basis of the followingdata: duration of mechanical ventilation, oxygen requirement at 28 dayswith an abnormal chest radiograph, oxygen requirement at 36 weeks PCA ordate of discharge.

Growth parameters were assessed at birth, 28 days of age and 36 weeksPCA, Blood chemistries and liver function tests were evaluated at theonset of the study and on days 7 and 28 post-administration. Completeblood counts and urinalysis were performed on enrollment, 24, 48 and 72h, 7 and 28 d post-administration. Cranial ultrasounds were performedupon randomization and were repeated at 7 and 28 d of life.

EXAMPLE 12 Incidence of PVL and IVH Adverse Events

There were no instances of PVL (peri-ventricular leukomalacia) in therhCC10-treated infants. However, there was one infant in the placebogroup who developed PVL. PVL occurs when leukocytes (primarilyneutrophils) infiltrate the brain and cause a severe inflammatoryresponse. PVL, if not lethal, typically results in severe neurologicalimpairment in the infant. Smaller and younger infants are predisposed toPVL. Even though the infants in the high dose group (5.0 mg/kg patientbody mass) were smaller and younger than in the placebo group, there wasno PVL in the high dose group. rhCC10 thus protected these disadvantagedinfants from PVL.

Likewise, the incidence of IVH (Intraventricular hemorrhage), whichoccurs when a blood vessel in the brain bursts in response to aggressiveventilation and high oxygen levels, was lower in the rhCC10 treatedgroups than in the placebo group. RhCC10 appears to have protected thesedisadvantaged infants in the high dose group from IVH, refuting thescientific papers that taught that rhCC10 inhibits platelet aggregationand would promote hemorrhaging in vivo.

PDA (patent ductus arteriosis), a defect or incomplete closure in thewalls of the heart, was significantly decreased in the rhCC10-treatedgroups compared to placebo. PDA is a life-threatening problem that mustbe corrected surgically, if it is not resolved in the first severalmonths of life. rhCC10 reduced the incidence of PDA, possibly bydecreasing the stress on the heart.

There were no significant differences in values obtained for bloodchemistries, complete blood counts or results of urinalysis among groupsat any of the time points evaluated.

There were no significant differences in the incidence ofnon-respiratory adverse events in the treatment and placebo groups(Table 2). Three cases of NEC occurred at one center in rhCC10-treatedinfants. However, other premature infants not enrolled in the study atthat center also developed NEC at the same time. Growth parameters weresimilar among the groups.

EXAMPLE 13 Immunological Safety

It will be further appreciated that the safety of rhCC10 can be measuredby conducting an analysis of the potential immunogenicity of theadministered rhCC10 using plasma samples. Plasma samples collected onday 28 of life were tested for the presence of anti-CC10 antibodies. Noevidence of antibody formation was present in any of the 28 day plasmasamples from any of the groups.

EXAMPLE 14 Other Outcomes

With reference to Table 2, there were no significant differences in theincidence of non-respiratory adverse events in the treatment and placebogroups. Three cases of NEC occurred at one center in rhCC0-treatedinfants. However, other premature infants not enrolled in the study atthat center also developed NEC at the same time. Growth parameters at 36weeks CGA were similar among the groups. Similarly, there were nosignificant differences in values obtained for blood chemistries,complete blood counts or results of urinalysis among groups at any ofthe time points evaluated. Thus, rhCC10 administration, in contrast tocorticosteroids, did not appear to cause any significant safety issuesin premature infants.

In addition, IT rhCC10 did not elicit an immunogenic response fromtreated infants. The only adverse event that was increased in treatedinfants compared to placebo controls was NEC (p=NS), however, it was notpossible to attribute the NEC to the administration of rhCC10 for tworeasons. First, excess CC10 was cleared from all infants by 48 hpost-administration and the 3 cases of confirmed NEC occurred 3-6 wkpost-administration. Second, all cases of confirmed NEC occurred at thesame center. In addition, there were other cases of NEC in infants notenrolled in the rhCC10 study in this center occurring in the sametimeframe, suggesting an outbreak pattern. These data indicate it ishighly unlikely that rhCC10 administration was related to thedevelopment of NEC.

Referring now to, for example, Examples 10-13, it has been shown thatrhCC10 is safe and well-tolerated because, upon administration to apatient, it does not elicit any immediate or delayed local or systemicreactions in the patient, it is not associated with any unusual adverseevents, or increased severity or frequency of typical adverse events forthe treated patient population, as described above. Furthermore, rhCC10is safe and well-tolerated because it does not elicit any immunologicresponse from the patient to either the rhCC10 or to endogenous CC10does not predispose the patient to bleeding or hemorrhage, orspecifically increase the platelet aggregation time in the patient, anddoes not compromise the patient's immune function and predispose thepatient to infection.

EXAMPLE 15

A patient, who may be an adult, child or a premature infant presentswith RDS, BDP, chronic lung disease (in the case of a child or anadult), pulmonary fibrosis (in the case of a child or an adult), asthma,or COPD. A dose of rhCC10 from 1.5 mg/kg to 5 mg/kg of patient body massis given to the patient. The patient will then demonstrate a resolutionof the symptoms of the aforementioned condition.

EXAMPLE 16

A patient, who may be an adult, child or a premature infant presentswith RDS, BDP, chronic lung disease (in the case of a child or anadult), pulmonary fibrosis (in the case of a child or an adult), asthma,or COPD. A dose of rhCC10 from 0.15 ng/kg to 5 mg/kg of patient bodymass is given to the patient. The patient will then be relieved of thesymptoms of the aforementioned condition.

EXAMPLE 17

A patient, who may be an adult, child or a premature infant presentswith RDS, BDP, chronic lung disease (in the case of a child or anadult), pulmonary fibrosis (in the case of a child or an adult), asthma,or COPD. A dose of rhCC10 from 0.15 mg/kg to 5 mg/kg of patient bodymass is given to the patient. The patient will then be relieved of thesymptoms of the aforementioned condition.

Based on the foregoing, the critical ranges for rhCC10 dosages effectiveto safely treat, cure and prevent RDS, BDP, chronic lung disease and/orpulmonary fibrosis, asthma, and COPD have been found. Accordingly, thepresent invention provides a safe and well-tolerated rhCC10 basedtherapy effective at treating the symptoms of RDS, BDP, chronic lungdisease and/or pulmonary fibrosis, asthma, and COPD thus increasing thelong term survivability of both premature infants, child and adultpatients suffering from these conditions, while not causing anydangerous side effects.

1. A method of decreasing white blood cell proliferation in a subjectcomprising the steps of: (a) diagnosing the subject with one or more ofrespiratory distress syndrome, broncho-pulmonary dysplasia, pulmonaryfibrosis and chronic obstructive pulmonary disorder; (b) collecting afirst blood sample; (c) determining a white blood cell count for thefirst sample of blood; (d) administering 1.5 mg to 5 mg rhCC10 per kg ofsubject body mass to a subject by the intratracheal, endotracheal,dialysate, ophthalmic, intravenous, systemic, or oral routes; (e)collecting a second blood sample; (f) determining a white blood cellcount for the second sample of blood; (g) observing a white blood cellcount in the second blood sample which is at least 2% lower than thewhite blood cell count in the first blood sample.
 2. The method of claim1 wherein neutrophil levels are determined for the first and secondblood samples and decreased by at least 2% after administration ofrhCC10.
 3. The method of claim 1 wherein polymorphonuclear leukocytelevels are determined for the first and second blood samples anddecreased by at least 2% after administration of rhCC10.
 4. A method ofdecreasing total cell counts in the tracheal aspirate fluids of asubject comprising the steps of: (a) diagnosing the subject with one ormore of respiratory distress syndrome, broncho-pulmonary dysplasia,pulmonary fibrosis and chronic obstructive pulmonary disorder; (b)collecting a first sample of tracheal aspirate fluid; (c) determining atotal cell count for the first sample of tracheal aspirate fluid; (d)administering 1.5 mg to 5 mg rhCC10 per kg of subject body mass to asubject by the intratracheal, endotracheal, dialysate, ophthalmic,intravenous, systemic, or oral routes; (e) collecting a second sample oftracheal aspirate fluid; (f) determining a total cell count for thesecond sample of tracheal aspirate fluid; (g) observing a total cellcount in the second sample of tracheal aspirate fluid which is at least2% lower than the total cell count in the first sample of trachealaspirate fluid.
 5. The method of claim 4 wherein the subject reaches apeak combined tracheal aspirate fluid concentration of native CC10 andrhCC10 between about 12 and about 48 hours after initial dosage andwherein the combined concentration of native CC10 and rhCC10 is measuredbetween about 12 and about 48 hours after initial dosage.
 6. The methodof claim 1 wherein the administration of rhCC10 is repeated about onceevery 48 hours.